Methods and devices for bone infection treatment selection

ABSTRACT

Disclosed are methods for identifying patients at increased risk of developing disseminated staphylococcal infection, which includes the steps of determining that the patient has a mutation in one or more genes selected from a MPDZ network gene, a CGNL1 network gene, a PRKRIR network gene, a MED26 network gene, a tight junction protein gene, or an immune modulator gene; and treating the patient for disseminated staphylococcal infection. Also disclosed are solid substrates and/or assays useful for carrying out the disclosed methods.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of and priority to U.S. patentapplication Ser. No. 15/978,332, filed May 14, 2018, entitled “Methodsand Devices for Bone Infection Treatment Selection,” which claims thebenefit of and priority to U.S. patent application Ser. No. 14/681,673,filed Apr. 8, 2015, entitled “Methods and Devices for Bone InfectionTreatment Selection,” issued as U.S. Pat. No. 9,999,652 on Jun. 19,2018, which claims the benefit of and priority to U.S. ProvisionalPatent Application Ser. No. 61/978,614, filed Apr. 11, 2014, entitled“Methods and Devices for Bone Infection Treatment Selection,” thecontents of which are incorporated herein in their entirety for allpurposes.

BACKGROUND

Osteomyelitis is the second most common invasive childhood infectionthat requires hospitalization; nevertheless, its pathogenesis is poorlyunderstood. Staphylococcus aureus is an ever-present pathogen for bothchildren and adults. Although skin and soft tissue infections are themost common reason for treatment, invasive disease due to S. aureus isalso on the rise: sepsis, pneumonia, osteomyelitis, septic arthritis,brain abscess. Osteomyelitis accounts for a substantial number of casesof invasive S. aureus disease. Osteomyelitis is thought to occur when S.aureus or other bacteria enter the skin, invade the bloodstream, and arecarried to the metaphysis of a long bone. Most children present withinfection in a single bone and improve quickly with 7-10 days ofintravenous antibiotics and another 2-3 weeks of oral therapy. Despiteencompassing knowledge of the organism and its virulence factors, verylittle is known about host susceptibility to S. aureus.

As such, there is a need for compositions and methods for detection ofindividuals susceptible to S. aureus, such that appropriate treatmentmay be administered. The instant disclosure seeks to address one or moreof these needs in the art.

SUMMARY

Disclosed are methods for identifying patients at increased risk ofdeveloping disseminated staphylococcal infection, which includes thesteps of determining that the patient has a mutation in one or moregenes selected from a MPDZ network gene, a CGNL1 network gene, a PRKRIRnetwork gene, a MED26 network gene, a tight junction protein gene, or animmune modulator gene, or combinations thereof; and treating the patientfor disseminated staphylococcal infection. Also disclosed are solidsubstrates and/or assays useful for carrying out the disclosed methods.

DETAILED DESCRIPTION

Disclosed herein are methods and compositions for the stratification ofpatients having bone infection, wherein patients having a need forsurgical intervention and/or specific antibiotics (e.g., bacteriocidalantibiotics) are identified.

The term “biomarker” as used herein refers to a gene that comprises oneor more mutations as compared to the wild type gene and is predictive ofdifferent disease prognosis, survival outcome, or preferred methods oftreatment.

The term “nucleic acid” includes DNA and RNA and can be either doublestranded or single stranded.

The term “hybridize” or “hybridizable” refers to the sequence specificnon-covalent binding interaction with a complementary nucleic acid.Hybridization may be under high stringency conditions. Appropriatestringency conditions which promote hybridization will be understood tothose skilled in the art.

The term “probe” as used herein refers to a nucleic acid sequence thatwill hybridize to a nucleic acid target sequence (e.g., a biomarker). Inone example, the probe hybridizes to an RNA product of the biomarker ora nucleic acid sequence complementary thereof. The length of probedepends on the hybridization conditions and the sequences of the probeand nucleic acid target sequence. In one embodiment, the probe may be atleast 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500 or morenucleotides in length.

A “region” of a probe or biomarker, as used herein, may comprise orconsist of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29 or more contiguous nucleotides from a particulargene or a complementary sequence thereof. In some embodiments, theregion may be of the same length as the probe or the biomarker. In otherembodiments, the region may be shorter than the length of the probe orthe biomarker.

The term “subject” as used herein refers to any member of the animalkingdom.

The term “target specific probe” encompasses probes that have a regionof contiguous nucleotides having a sequence that is either (i)identically present in one of the disclosed genes, or (ii) complementaryto the sequence of a region of contiguous nucleotides found in one ofthe disclosed genes, where “region” can comprise the full lengthsequence of any one of the disclosed genes, a complementary sequence ofthe full length sequence of any one of the disclosed genes, or asubsequence thereof.

Applicant has identified mutations in certain genes that may operate asbiomarkers that can be used to screen subjects with osteomyelitis, inparticular children, immediately upon diagnosis. The biomarkers may beused in conjunction with a solid surface array as is known in the art.Those subjects with mutations in the certain genes (the biomarkers) maythen be considered at risk for disseminated staphylococcal infectionafter osteomyelitis and would receive bactericidal antibiotics withsurgical extirpation of the site of infection in bone. A solid surfacearray or other screening device, as described herein or as otherwiseappreciated in the art, may be used as a risk-stratification tool topredict which subjects with osteomyelitis have an underlying geneticsusceptibility for disseminated staphylococcal infection as aconsequence of osteomyelitis, such that they may be quickly treatedusing the most effective therapeutic protocol.

Method of Treatment

In one aspect, a method of treatment is disclosed. The method maycomprise the steps of a) identifying a patient having a bone infection;b) determining that the patient has a mutation in one or more genesselected from a MPDZ network gene, a CGNL1 network gene, a PRKRIRnetwork gene, a MED26 network gene, a tight junction protein gene, animmune modulator gene, and combinations thereof; and c) treating thepatient for disseminated staphylococcal infection.

In one aspect, the bone infection may be osteomyelitis.

In one aspect, the treatment step may comprise surgical intervention,treatment with a bactericidal antibiotic, or a combination thereof.

In one aspect, the treatment step may comprise surgical intervention,treatment with a bactericidal antibiotic for Staphylococcus aureus, or acombination thereof.

In one aspect, the antibiotic used in the treatment step may be selectedfrom teicoplanin, vancomycin, gentamicin, and combinations thereof. Inone aspect, the antibiotic may comprise vancomycin.

In one aspect, the gene may be selected from MPDZ, CLDN1, CLDN4, CLDN5,CLDN8, TLN2, KCNJ15, SYNGP1, PLEKHA2, AMOT, CGNL1, CGN, TMOD3, TMOD2,DMXL2, TLN2, MPAK6, DOK4, CDH5, GATA4, GEF-H1, TIAM1, PRKRIR, STK4,DNAJC3, EIF2AK2, EIF2 S1, DDX58, TP53, UBC, MED1,4, MED6,7,8,9, MED10,11, 12, MED 13, 14, 15, MED 17, 18, 19, MED20, 21, 23, MED25, 26, 27,28, MED 29, 30, 31, SREBF1, TRRAP, CTDP1, TADA2A, CDK8, 19, Z01,2,3,CTNNA1, 2, 3, CTNNB1, MARVELD2, 3, OCLN, JAM2, 3, ESAM, CYBA, CYBB,NCF2, CEBPE, STAT1, IL1R1, TNFSF11, IL12RB1, IFNGR1/2, and combinationsthereof (See Table 1).

TABLE 1 MPDZ CGNL1 PRKRIR MED26 Tight junction Immune network (10)network (12) network (8) Network (31) proteins (13) modulators (9) MPDZCGNL1 PRKRIR MED1, 4 ZO1, 2, 3 CYBA CLDN1 CGN STK4 MED6, 7, 8, 9 CTNNA1,2, 3 CYBB CLDN4 TMOD3 DNAJC3 MED10, 11, 12 CTNNB1 NCF2 CLDN5 TMOD2EIF2AK2 MED13, 14, 15 MARVELD2, 3 CEBPE CLDN8 DMXL2 EIF2S1 MED17, 18, 19OCLN STAT1 TLN2 TLN2 DDX58 MED20, 21, 23 JAM2, 3 IL1R1 KCNJ15 MPAK6 TP53MED25, 26, 27, 28 ESAM TNFSF11 SYNGAP1 DOK4 UBC MED29, 30, 31 IL12RB1PLEKHA2 CDH5 SREBF1 IFNGR1/2 AMOT GATA4 TRRAP GEF-H1 CTDP1 TIAM1 TADA2ACDK8, 19

TABLE 2 Genes and representative, non-exhaustive, accession numbers.MPDZ NM_003829.4 MED17 NM_004268.4 CLDN1 NM_021101.4 MED18 NM_017638.2CLDN4 NM_001305.4 MED19 NM_153450.1 CLDN5 NM_001130861.1 MED20NM_004275.3 CLDN8 NM_199328.2 MED21 NM_004264.4 TLN2 NM_015059.2 MED23NM_004830.3 SYNGAP1 NM_006772.2 MED25 NM_030973.3 PLEKHA2 NM_021623.1MED26 NM_004831.3 AMOT NM_001113490.1 MED27 NM_004269.3 KCNJ15NM_170736.2 MED28 NM_025205.3 CGNL1 NM_001252335.1 MED29 NM_017592.1 CGNNM_020770.2 MED30 NM_080651.3 TMOD3 NM_014547.4 MED31 NM_016060.2 TMOD2NM_014548.3 SREBF1 NM_001005291.2 DMXL2 NM_001174116.1 TRRAPNM_001244580.1 MPAK6 CTDP1 NM_004715.4 DOK4 NM_018110.3 TADA2ANM_001488.3 CDH5 NM_001795.3 CDK8 NM_001260.1 GATA4 NM_002052.3 CDK19NM_015076.3 GEF-H1 NM_001162383 ZO1 TIAM1 NM_003253.2 ZO2 PRKRIRNM_004705.2 ZO3 STK4 NM_006282.2 CTNNA1 NM_001903.3 DNAJC3 NM_006260.4CTNNA2 NM_004389.3 EIF2AK2 NM_001135652.2 CTNNA3 NM_013266.3 EIF2S1NM_004094.4 CTNNB1 NM_001904.3 DDX58 NM_014314.3 MARVELD2 NM_001038603.2TP53 M_000546.5 MARVELD3 NM_001017967.3 UBC NM_021009.6 OCLN NM_002538.3MED1 NM_004774.3 JAM2 NM_021219.3 MED4 NM_014166.3 JAM3 NM_032801.4 MED6NM_001284209.1 ESAM NM_138961.2 MED7 NM_001100816.1 CYBA NM_000101.3MED8 NM_001001653.2 CYBB NM_000397.3 MED9 NM_018019.2 NCF2 NM_000433.3MED10 NM_032286.2 CEBPE NM_001805.3 MED11 NM_001001683.2 STAT1NM_007315.3 MED12 NM_005120.2 IL1R1 NM_000877.3 MED15 NM_001003891.1TNFSF11 NM_003701.3 MED14 NM_004229.3 IL12RB1 NM_005535.2 MED13NM_005121.2 IFNGR1 NM_000416.2 IFNGR2 NM_005534.3

The above list of accession numbers is intended to be for referenceonly, and is not intended to limit the scope of the invention. The fullsequence as listed in the accession database for the above-referencednumbers is incorporated herein by reference. It is understood thatvariants of the disclosed genes are within the scope of the invention,as would be understood by one of ordinary skill in the art.

In one aspect, the mutation may be selected from a mutation in a PDZdomain of MPDZ, a mutation in an L27-2 domain of MPDZ, a Leu35Promutation in MPDZ, a mutation in a rod domain of CGNL1, a Leu to Ilemutation in CGNL1, a Leu739Ile mutation in CGNL1, a mutation in anuclear export domain of PRKRIR, a Leu602Phe mutation of PRKRIR, amutation in a TFIIS domain of MED26, an Ala464Thr mutation in MED26, orcombinations thereof.

Arrays

In one aspect, an article for detecting one or more mutations in a genesequence comprising a) a solid support; and b) one or more probes forassessing the presence or absence of a mutation in a gene sequence,wherein said gene may be selected from a MPDZ network gene, a CGNL1network gene, a PRKRIR network gene, a MED26 network gene, a tightjunction protein gene, an immune modulator gene, and combinationsthereof; wherein said one or more probes may be affixed to said solidsupport; and wherein said one or more probes overlaps a regioncomprising said mutation in a gene sequence.

In one aspect, the MPDZ network may comprise MPDZ, CLDN1, CLDN4, CLDN5,CLDN8, TLN2, KCNJ15, SYNGP1, PLEKHA2, AMOT, and combinations thereof.

In one aspect, the CGNL1 network may comprise CGNL1, CGN, TMOD3, TMOD2,DMXL2, TLN2, MPAK6, DOK4, CDH5, GATA4, GEF-H1, TIAM1, and combinationsthereof.

In one aspect, the PRKRIR network may comprise PRKRIR, STK4, DNAJC3,EIF2AK2, EIF2S1, DDX58, TP53, UBC, and combinations thereof.

In one aspect, the MED26 network may comprise MED1,4, MED6,7,8,9, MED10,11, 12, MED 13, 14, 15, MED 17, 18, 19, MED20, 21, 23, MED25, 26, 27,28, MED 29, 30, 31, SREBF1, TRRAP, CTDP1, TADA2A, CDK8, 19, andcombinations thereof.

In one aspect, the one or more sequences encoding a tight junctionprotein may comprise Z01,2,3, CTNNA1, 2, 3, CTNNB1, MARVELD2, 3, OCLN,JAM2, 3, ESAM, and combinations thereof.

In one aspect, the one or more sequences encoding an immune modulatormay comprise CYBA, CYBB, NCF2, CEBPE, STAT1, IL1R1, TNFSF11, IL12RB1,IFNGR1/2, and combinations thereof.

In one aspect, the solid support may comprise the MPDZ sequence.

In one aspect, the solid support may comprise the MPDZ sequence, whereinsaid MPDZ sequence may comprise a mutation in a PDZ domain.

In one aspect, the solid support may comprise the MPDZ sequence, whereinsaid MPDZ sequence may comprise a mutation in an L27-2 domain.

In one aspect, the solid support may comprise the MPDZ sequence, whereinsaid sequence may comprise a Leu35Pro mutation.

In one aspect, the solid support may comprise the CGNL1 sequence.

In one aspect, the solid support may comprise the CGNL1 sequence,wherein said CGNL1 sequence may comprise a mutation in a rod domain.

In one aspect, the solid support may comprise the CGNL1 sequence,wherein said CGNL1 sequence may comprise a Leu to Ile mutation.

In one aspect, the solid support may comprise the CGNL1 sequence,wherein said CGNL1 sequence may comprise a Leu739Ile mutation.

In one aspect, the solid support may comprise the PRKRIR sequence.

In one aspect, the solid support may comprise the PRKRIR sequence,wherein said PRKRIR sequence may comprise a mutation in a nuclear exportdomain.

In one aspect, the solid support may comprise the PRKRIR sequence,wherein said PRKRIR sequence may comprise a Leu602Phe mutation.

In one aspect, the solid support may comprise the MED26 sequence.

In one aspect, the solid support may comprise the MED26 sequence,wherein said MED26 sequence may comprise a mutation in a TFIIS domain.

In one aspect, the solid support may comprise the MED26 sequence,wherein said MED26 sequence may comprise an Ala464Thr mutation.

A person skilled in the art will appreciate that a number of methods canbe used to detect a mutation in a sample, including arrays, such asmicroarrays, RT-PCR, nuclease protection assays, multiplex assaysincluding nanostring technology, and Northern blot analyses. Anyanalytical procedure capable of permitting specific and quantifiable (orsemi-quantifiable) detection of the biomarkers may be used in thedisclosed methods, such as the microarray and quantitative PCR, e.g.quantitative RT-PCR, methods set forth herein, and methods known tothose skilled in the art.

Accordingly, in one aspect, the mutation may be determined using arrays,optionally microarrays, RT-PCR, optionally quantitative RT-PCR, nucleaseprotection assays or Northern blot analyses.

In some aspects, the mutation may be determined by using an array. cDNAmicroarrays may consist of multiple (usually thousands) of differentcDNA probes spotted (usually using a robotic spotting device) onto knownlocations on a solid support, such as a glass microscope slide. Sucharrays are well known in the art. Microarrays for use in the methodsdescribed herein may comprise a solid substrate onto which the probesare covalently or non-covalently attached. The cDNAs are typicallyobtained by PCR amplification of plasmid library inserts using primerscomplementary to the vector backbone portion of the plasmid or to thegene itself for genes where sequence is known. PCR products suitable forproduction of microarrays are typically between 0.5 and 2.5 kB inlength. In a typical microarray experiment, RNA (either total RNA orpoly A RNA) may be isolated from cells or tissues of interest and isreverse transcribed to yield cDNA. Labeling is usually performed duringreverse transcription by incorporating a labeled nucleotide in thereaction mixture. A microarray may then be hybridized with labeled RNA.Microarray analysis can be performed by commercially availableequipment, following manufacturer's protocols, such as by usingAffymetrix GeneChip technology, Agilent Technologies cDNA microarrays,Illumina Whole-Genome DASL array assays, or any other comparablemicroarray technology.

In some aspects, probes capable of hybridizing to one or more RNAs orcDNAs may be attached to a substrate at a defined location (“addressablearray”). Probes can be attached to the substrate in a wide variety ofways, as will be appreciated by those in the art. In some embodiments,the probes are synthesized first and subsequently attached to thesubstrate. In other embodiments, the probes are synthesized on thesubstrate. In some aspects, probes may be synthesized on the substratesurface using techniques such as photopolymerization andphotolithography.

In some aspects, microarrays may be utilized in a RNA-primed,Array-based Klenow Enzyme (“RAKE”) assay. See Nelson, P. T. et al.(2004) Nature Methods 1(2):1-7; Nelson, P. T. et al. (2006) RNA12(2):1-5. In these embodiments, total RNA may be isolated from asample. Optionally, small RNAs can be further purified from the totalRNA sample. The RNA sample is then hybridized to DNA probes immobilizedat the 5′-end on an addressable array. The DNA probes may comprise abase sequence that is complementary to a target RNA of interest, such asone or more RNAs capable of specifically hybridizing to a nucleic acidcomprising a sequence identified herein.

In some aspects, the array may comprise DNA probes for no more than thegenes disclosed herein. In other aspects, the array may compriseadditional DNA probes.

In one aspect, after the sample is hybridized to the array, the samplemay be exposed to exonuclease I to digest any unhybridized probes. TheKlenow fragment of DNA polymerase I may then be applied along withbiotinylated dATP, allowing the hybridized biomarker to act as primersfor the enzyme with the DNA probe as template. The slide may then bewashed and a streptavidin-conjugated fluorophore may be applied todetect and quantify the spots on the array containing hybridized andKlenow-extended biomarker RNAs from the sample.

In some embodiments, an RNA sample may be reverse transcribed using abiotin/poly-dA random octamer primer. The RNA template may be digestedand the biotin-containing cDNA may be hybridized to an microarray withbound probes that permit specific detection of biomarker RNAs. Themicroarray may include at least one probe comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19,even at least 20, 21, 22, 23, or 24 contiguous nucleotides identicallypresent in each of the genes disclosed herein. After hybridization ofthe cDNA to the microarray, the microarray may then be exposed to astreptavidin-bound detectable marker, such as a fluorescent dye, and thebound cDNA is detected, as is known in the art.

In some aspects, compositions are provided that comprise at least onetarget specific probe.

In some aspects, target specific probes may consist ofdeoxyribonucleotides. In other aspects, target specific probes mayconsist of both deoxyribonucleotides and nucleotide analogs. In someaspects, biomarker RNA-specific probes comprise at least one nucleotideanalog which may increase the hybridization binding energy. In someaspects, a target specific probe in the compositions described hereinbinds to one biomarker RNA or DNA in the sample.

In some aspects, more than one probe specific for a single biomarkersequence may be present in the compositions, the probes capable ofbinding to overlapping or spatially separated regions of the biomarkersequence.

It will be understood that in some embodiments in which the compositionsdescribed herein are designed to hybridize to cDNAs reverse transcribedfrom biomarker RNAs, the composition may comprise at least one targetspecific probe comprising a sequence that is identically present in abiomarker RNA (or a subsequence thereof).

In some aspects, a biomarker sequence may be capable of specificallyhybridizing to at least one probe comprising a base sequence that isidentically present in one of the genes disclosed herein. In someaspects, a biomarker sequence may be capable of specifically hybridizingto at least one nucleic acid probe comprising a sequence that isidentically present in one of the genes disclosed herein.

In some aspects, the composition may comprise a plurality of target orbiomarker sequence specific probes each comprising a region ofcontiguous nucleotides comprising a base sequence that is identicallypresent in one or more of the disclosed genes, or in a subsequence of adisclosed gene. In some aspects, the composition may comprise aplurality of target or biomarker sequence specific probes eachcomprising a region of contiguous nucleotides comprising a base sequencethat is complementary to a sequence of a disclosed gene.

In some aspects, the microarray may comprise probes comprising a regionwith a base sequence that is fully complementary to a target region of abiomarker. In other aspects, the microarray may comprise probescomprising a region with a base sequence that may comprise one or morebase mismatches when compared to the sequence of the best-aligned targetregion of a biomarker.

In some aspects, the microarray may comprise may comprise probes eachcomprising a region of at least 10 contiguous nucleotides, such as atleast 11 contiguous nucleotides, such as at least 13 contiguousnucleotides, such as at least 14 contiguous nucleotides, such as atleast 15 contiguous nucleotides, such as at least 16 contiguousnucleotides, such as at least 17 contiguous nucleotides, such as atleast 18 contiguous nucleotides, such as at least 19 contiguousnucleotides, such as at least 20 contiguous nucleotides, such as atleast 21 contiguous nucleotides, such as at least 22 contiguousnucleotides, such as at least 23 contiguous nucleotides, such as atleast 24 contiguous nucleotides, such as at least 25 contiguousnucleotides with a base sequence that is identically present in one ofthe disclosed genes, or a variant thereof.

In some aspects, the microarray component may comprise probes eachcomprising a region with a base sequence that is identically present ineach of the disclosed genes, or a variant thereof.

In some aspects, the analytical method used for detecting at least onebiomarker may include real-time quantitative RT-PCR. Although PCR canuse a variety of thermostable DNA-dependent DNA polymerases, ittypically employs the Taq DNA polymerase, which has a 5′-3′ nucleaseactivity but lacks a 3′-5′ proofreading endonuclease activity. In someaspects, RT-PCR is done using a TaqMan® assay sold by AppliedBiosystems, Inc. RT-PCR may be performed using any RT-PCRinstrumentation available in the art. Instrumentation used in real-timeRT-PCR data collection and analysis may comprise a thermal cycler,optics for fluorescence excitation and emission collection, andoptionally a computer and data acquisition and analysis software.

In various other aspects, RT-PCR detection may be utilized to detect, ina single multiplex reaction, one or more biomarkers. The biomarkers, insome aspects, may be capable of specifically hybridizing to a nucleicacid comprising a sequence that is identically present in one of thedisclosed genes.

In some aspects, a plurality of probes, such as TaqMan probes, eachspecific for a different target sequence, may be used. Eachtarget-specific probe may be spectrally distinguishable from the otherprobes used in the same multiplex reaction.

In some aspects, the methods of detecting at least one biomarkerdescribed herein may employ one or more modified oligonucleotides, suchas oligonucleotides comprising one or more affinity-enhancingnucleotides. Modified oligonucleotides useful may include primers forreverse transcription, PCR amplification primers, and probes. In someaspects, the incorporation of affinity-enhancing nucleotides mayincreases the binding affinity and specificity of an oligonucleotide forits target nucleic acid as compared to oligonucleotides that containonly deoxyribonucleotides, and allows for the use of shorteroligonucleotides or for shorter regions of complementarity between theoligonucleotide and the target nucleic acid. Affinity-enhancingnucleotides may include nucleotides comprising one or more basemodifications, sugar modifications and/or backbone modifications.Modified bases for use in affinity-enhancing nucleotides may include5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,2-chloro-6-aminopurine, xanthine and hypoxanthine. Affinity-enhancingmodifications may include nucleotides having modified sugars such as2′-substituted sugars, such as 2′-O-alkyl-ribose sugars,2′-amino-deoxyribose sugars, 2′-fluoro-deoxyribose sugars,2′-fluoro-arabinose sugars, and 2′-O-methoxyethyl-ribose (2′MOE) sugars.In some aspects, modified sugars are arabinose sugars, ord-arabino-hexitol sugars. Affinity-enhancing modifications may alsoinclude backbone modifications such as the use of peptide nucleic acids(e.g., an oligomer including nucleobases linked together by an aminoacid backbone). Other backbone modifications include phosphorothioatelinkages, phosphodiester modified nucleic acids, combinations ofphosphodiester and phosphorothioate nucleic acid, methylphosphonate,alkylphosphonates, phosphate esters, alkylphosphonothioates,phosphoramidates, carbamates, carbonates, phosphate triesters,acetamidates, carboxymethyl esters, methylphosphorothioate,phosphorodithioate, p-ethoxy, and combinations thereof.

In some aspects, the oligomer may include at least oneaffinity-enhancing nucleotide that has a modified base, at leastnucleotide (which may be the same nucleotide) that has a modified sugar,and at least one internucleotide linkage that is non-naturallyoccurring. In some aspects, the affinity-enhancing nucleotide maycontain a locked nucleic acid (“LNA”) sugar, which is a bicyclic sugar.In some aspects, an oligonucleotide for use in the methods describedherein may comprise one or more nucleotides having an LNA sugar. In someaspects, the oligonucleotide contains one or more regions consisting ofnucleotides with LNA sugars. In other aspects, the oligonucleotidecontains nucleotides with LNA sugars interspersed withdeoxyribonucleotides. See, e.g., Frieden, M. et al. (2008) Curr. Pharm.Des. 14(11):1138-1142.

REFERENCES

-   1. Ardura M I, Banchereau R, Mejias A, Di Pucchio T, Glaser C,    Allantaz F, Pascual V, Banchereau J, Chaussabel D, Ramilo O.    Enhanced monocyte response and decreased central memory T cells in    children with invasive Staphylococcus aureus infections. PLoS One    2009; 4(5):e5446. PMCID: 2676512.-   2. Holland S M, DeLeo F R, Elloumi H Z, Hsu A P, Uzel G, Brodsky N,    Freeman A F, Demidowich A, Davis J, Turner M L, Anderson V L,    Darnell D N, Welch P A, Kuhns D B, Frucht D M, Malech H L, Gallin J    I, Kobayashi S D, Whitney A R, Voyich J M, Musser J M, Woellner C,    Schaffer A A, Puck J M, Grimbacher B. STAT3 mutations in the    hyper-IgE syndrome. N Engl J Med 2007; 357(16):1608-19.-   3. Ku C L, Picard C, Erdos M, Jeurissen A, Bustamante J, Puel A, von    Bernuth H, Filipe-Santos O, Chang H H, Lawrence T, Raes M, Marodi L,    Bossuyt X, Casanova J L. IRAK4 and NEMO mutations in otherwise    healthy children with recurrent invasive pneumococcal disease. J Med    Genet 2007; 44(1):16-23. PMCID: 2597905.-   4. Dinauer M C. Chronic granulomatous disease and other disorders of    phagocyte function. Hematology Am Soc Hematol Educ Program    2005:89-95.-   5. Vissers L E, de Ligt J, Gilissen C, Janssen I, Steehouwer M, de    Vries P, van Lier B, Arts P, Wieskamp N, del Rosario M, van Bon B W,    Hoischen A, de Vries B B, Brunner H G, Veltman J A. A de novo    paradigm for mental retardation. Nat Genet 2010; 42(12):1109-12.-   6. Le Saux N, Howard A, Barrowman N J, Gaboury I, Sampson M,    Moher D. Shorter courses of parenteral antibiotic therapy do not    appear to influence response rates for children with acute    hematogenous osteomyelitis: a systematic review. BMC infectious    diseases 2002; 2:16. PMCID: 128824.-   7. Pulimeno P, Paschoud S, Citi S. A role for ZO-1 and PLEKHA7 in    recruiting paracingulin to tight and adherens junctions of    epithelial cells. J Biol Chem 2011; 286(19):16743-50. PMCID:    3089516.-   8. Paschoud S, Yu D, Pulimeno P, Jond L, Turner J R, Citi S.    Cingulin and paracingulin show similar dynamic behaviour, but are    recruited independently to junctions. Molecular membrane biology    2011; 28(2):123-35.-   9. Wongdee K, Pandaranandaka J, Teerapornpuntakit J, Tudpor K,    Thongbunchoo J, Thongon N, Jantarajit W, Krishnamra N,    Charoenphandhu N. Osteoblasts express claudins and tight    junction-associated proteins. Histochemistry and cell biology 2008;    130(1):79-90.-   10. Guillemot L, Paschoud S, Jond L, Foglia A, Citi S. Paracingulin    regulates the activity of Racl and RhoA GTPases by recruiting Tiaml    and GEF-H1 to epithelial junctions. Molecular biology of the cell    2008; 19(10):4442-53. PMCID: 2555940.-   11. Paschoud S, Guillemot L, Citi S. Distinct domains of    paracingulin are involved in its targeting to the actin cytoskeleton    and regulation of apical junction assembly. J Biol Chem 2012;    287(16):13159-69. PMCID: 3340007.-   12. Prodromou C, Nuttall J M, Millson S H, Roe S M, Sim T S, Tan D,    Workman P, Pearl L H, Piper P W. Structural basis of the radicicol    resistance displayed by a fungal hsp90. ACS chemical biology 2009;    4(4):289-97.-   13. Tyrrell C, De Cecco M, Reynolds N L, Kilanowski F, Campopiano D,    Barran P, Macmillan D, Dorin J R. Isoleucine/leucine2 is essential    for chemoattractant activity of beta-defensin Defb14 through    chemokine receptor 6. Molecular immunology 2010; 47(6):1378-82.-   14. Takahashi H, Parmely T J, Sato S, Tomomori-Sato C, Banks C A,    Kong S E, Szutorisz H, Swanson S K, Martin-Brown S, Washburn M P,    Florens L, Seidel C W, Lin C, Smith E R, Shilatifard A, Conaway R C,    Conaway J W. Human mediator subunit MED26 functions as a docking    site for transcription elongation factors. Cell 2011; 146(1):92-104.    PMCID: 3145325.-   15. Tsutsui T, Fukasawa R, Shinmyouzu K, Nakagawa R, Tobe K, Tanaka    A, Ohkuma Y. Mediator complex recruits epigenetic regulators via its    two cyclin-dependent kinase subunits to repress transcription of    immune response genes. J Biol Chem 2013; 288(29):20955-65. PMCID:    3774365.-   16. Hoskins E E, Morris T A, Higginbotham J M, Spardy N, Cha E,    Kelly P, Williams D A, Wikenheiser-Brokamp K A, Duensing S, Wells    S I. Fanconi anemia deficiency stimulates HPV-associated    hyperplastic growth in organotypic epithelial raft culture. Oncogene    2009; 28(5):674-85. PMCID: 2636855.-   17. Lanaspa M A, Almeida N E, Andres-Hernando A, Rivard C J, Capasso    J M, Berl T. The tight junction protein, MUPP1, is up-regulated by    hypertonicity and is important in the osmotic stress response in    kidney cells. Proceedings of the National Academy of Sciences of the    United States of America 2007; 104(34):13672-7. PMCID: 1959440.-   18. Ohnishi H, Nakahara T, Furuse K, Sasaki H, Tsukita S, Furuse M.    JACOP, a novel plaque protein localizing at the apical junctional    complex with sequence similarity to cingulin. J Biol Chem 2004;    279(44):46014-22.-   19. Becamel C, Figge A, Poliak S, Dumuis A, Peles E, Bockaert J,    Lubbert H, Ullmer C. Interaction of serotonin 5-hydroxytryptamine    type 2C receptors with PDZ10 of the multi-PDZ domain protein MUPP1.    J Biol Chem 2001; 276(16):12974-82.-   20. Frank C F, Hostetter M K. Cleavage of E-cadherin: a mechanism    for disruption of the intestinal epithelial barrier by Candida    albicans. Translational research: the journal of laboratory and    clinical medicine 2007; 149(4):211-22.-   21. Bill B R, Petzold A M, Clark K J, Schimmenti L A, Ekker S C. A    primer for morpholino use in zebrafish. Zebrafish 2009; 6(1):69-77.    PMCID: 2776066.-   22. Sumanas S, Larson J D. Morpholino phosphorodiamidate    oligonucleotides in zebrafish: a recipe for functional genomics?    Briefings in functional genomics & proteomics 2002; 1(3):239-56.-   23. Sumanas S, Larson J D, Miller Bever M. Zebrafish chaperone    protein GP96 is required for otolith formation during ear    development. Developmental biology 2003; 261(2):443-55.-   24. Sumanas S, Lin S. Etsl-related protein is a key regulator of    vasculogenesis in zebrafish. PLoS biology 2006; 4(1):e10. PMCID:    1310653.-   25. Sumanas S, Zhang B, Dai R, Lin S. 15-zinc finger protein Bloody    Fingers is required for zebrafish morphogenetic movements during    neurulation. Developmental biology 2005; 283(1):85-96.-   26. Kwong R W, Perry S F. The tight junction protein claudin-b    regulates epithelial permeability and sodium handling in larval    zebrafish, Danio rerio. American journal of physiology Regulatory,    integrative and comparative physiology 2013; 304(7):R504-13. PMCID:    3627946.-   27. Collins M M, Baumholtz A I, Ryan A K. Claudin-5 expression in    the vasculature of the developing chick embryo. Gene expression    patterns: GEP 2012.-   28. Furuse M, Moriwaki K. The role of claudin-based tight junctions    in morphogenesis. Annals of the New York Academy of Sciences 2009;    1165:58-61.-   29. Gordon D L, Johnson G M, Hostetter M K. Ligand-receptor    interactions in the phagocytosis of virulent Streptococcus    pneumoniae by polymorphonuclear leukocytes. J Infect Dis 1986;    154(4):619-26.

It should be understood that every maximum numerical limitation giventhroughout this specification includes every lower numerical limitation,as if such lower numerical limitations were expressly written herein.Every minimum numerical limitation given throughout this specificationwill include every higher numerical limitation, as if such highernumerical limitations were expressly written herein. Every numericalrange given throughout this specification will include every narrowernumerical range that falls within such broader numerical range, as ifsuch narrower numerical ranges were all expressly written herein.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “20 mm” is intended to mean“about 20 mm.”

Every document cited herein, including any cross referenced or relatedpatent or application, is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed:
 1. A method of detecting a mutation in a humandiagnosed with a bone infection, said method comprising a) obtaining abiological sample from said human; and b) detecting whether a Leu739Ilemutation in CGNL1 is present in said biological sample with a probespecific for the Leu739Ile mutation in CGNL1 and detecting bindingbetween the probe and the Leu739Ile mutation in CGNL1.
 2. The method ofclaim 1 further comprising the step of administering an antibiotic tosaid human diagnosed with a bone infection, wherein said antibiotic isselected from teicoplanin, vancomycin, gentamicin, and combinationsthereof.
 3. The method of claim 1, further comprising the step ofdetecting whether a Leu602Phe mutation in PKKRIR is present in saidbiological sample using a nucleic acid probe comprising a detectablelabel and which is specific to said mutation.
 4. The method of claim 1,further comprising the step of detecting whether a Leu35Pro mutation inMPDZ is present using a nucleic acid probe comprising a detectable labeland specific to said mutation.
 5. The method of claim 1, furthercomprising the step of detecting whether a Ala464Thr mutation in MED26is present using a nucleic acid probe comprising a detectable label andspecific to said mutation.
 6. A method of detecting a mutation in ahuman diagnosed with a bone infection, said method comprising detectingthe presence of a Leu739Ile mutation in CGNL1 in a biological sampleobtained from said human with a probe specific for the Leu739Ilemutation; wherein said detecting step is carried out by detectingbinding between said probe and said Leu739Ile mutation in CGNL1.
 7. Themethod of claim 6, further comprising the step of detecting whether aLeu602Phe mutation in PKKRIR is present using a nucleic acid probecomprising a detectable label and specific to said mutation.
 8. Themethod of claim 6, further comprising the step of detecting whether aLeu35Pro mutation in MPDZ is present using a nucleic acid probecomprising a detectable label and specific to said mutation.
 9. Themethod of claim 6, further comprising the step of detecting whether aAla464Thr mutation in MED26 is present using a nucleic acid probecomprising a detectable label and specific to said mutation.